FISH Basic Method
- Each probe is specific to one region of a chromosome (pair), and is
labeled with fluorescent molecules throughout it's length.
- Each microscope slide contains many metaphases.
- Each metaphase consists of the complete set of chromosomes, one small
segment of which each probe will seek out and bind itself to.
The first step is to break apart (denature) the double strands of DNA
in both the probe DNA and the chromosome DNA so they can bind to each other.
This is done by heating the DNA in a solution of formamide at a high temperature
(70 C)
Next, the probe is placed on the slide and a glass coverslip is placed
on top. To prevent evaporation, the edges of the coverslip are sealed with
rubber cement. The slide is then placed in a 37 C incubator overnight for
the probe to hybridize with the target chromosome.
Overnight, the probe DNA seeks out it's target sequence on the specific
chromosome and binds to it. The strands slowly rejoin (reanneal).
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Main FISH page
University of Wisconsin - Madison
Waisman Center Cytogenetics Lab
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